UNIVERSIDAD AUTÓNOMA DE MADRID FACULTAD DE CIENCIAS DEPARTAMENTO DE BIOLOGÍA MOLECULAR Modeling Aurora B function and regulation TESIS DOCTORAL Gonzalo Fernández-Miranda Pérez MADRID, 2009 Ignacio Pérez de Castro Insúa, Investigador del Grupo de División Celular y Cáncer del Centro Nacional de Investigaciones Oncológicas (CNIO) y Marcos Malumbres Martínez, Jefe del Grupo de División Celular y Cáncer del Centro Nacional de Investigaciones Oncológicas (CNIO) Certifican: que Gonzalo Fernández-Miranda Pérez ha realizado bajo su dirección el trabajo de Tesis Doctoral titulado: Modeling Aurora B function and regulation Revisado el presente trabajo, consideran que reúne todos los méritos necesarios para su presentación y defensa con el fin de optar al grado de Doctor por la Universidad Autónoma de Madrid. Ignacio Pérez de Castro Marcos Malumbres Abbreviations Abbreviations 3-MC: 3-Methylcholanthrene Ad-CRE: Adenoviruses expressing CRE recombinase Ad-GFP: Adenoviruses expressing GFP BAC: Bacterial Artificial Chromosomes BrdU: Bromodeoxiuridine BSA: Bovine Serum Albumin cDNA: Commplementary DNA CMV: Cytomegalovirus promoter CNIO: Centro Nacional de Investigaciones Oncológicas DMBA: 7,12-dimethylbenz[]anthracene DMEM: Dulbecco‟s Modified Eagle‟s Medium DMSO: Dimethyl sulfoxide EDTA: Ethylenediaminetetraacetic Acid ES cells: Embryonic Stem Cells EV: Empty Vector FBS: Fetal Bovine Serum GAPDH: Glyceraldehyde-3-phosphate dehydrogenase GFP: Green Fluorescence Protein H&E: Haematoxylin and Eosin HR: Homologous Recombinantion IF: Immunofluorescence IHQ: Immunohistochemistry KDa: KiloDalton KO: Animals or cells derived from a gene knock-out strain KI: Animals or cells derived from a gene knock-in strain LacZ: beta-galactosidase MEFs: Mouse Embryonic Fibroblasts MMTV: Mouse mammary tumour virus promoter NEO: Neomicine PBS: Phosphate Buffered Saline PCR: Polymerase Chain Reaction PFA: Paraformaldehyde i Abbreviations qRT-PCR: quantitative Reverse Transcriptase Polymerase Chain Reaction rtTA: reverse tetracycline Transactivator SDS: Sodium Dodecyl Sulphate shRNA: short hairpin RNA siRNA: small interference RNA Tet: Tetracycline TetO: Tetracycline Operator elements Tet-P: Tetracycline-responsive minimal promoter Tg: Transgenic mice TK: Thymidine Kinase TPA: 12-O-tetradecanoylphorbol-13-acetate tTA: tetracycline Transactivator UAM: Universidad Autónoma de Madrid UTR: Untranslated region WB: Western-Blot WT: Wild Type X-GAL: 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside ii Index SUMMARY ...................................................................................................................................... 1 RESUMEN ........................................................................................................................................ 3 INTRODUCTION ............................................................................................................................ 5 1. THE MAMMALIAN CELL DIVISION CYCLE ..................................................................................... 5 2. CELL CYCLE CHECKPOINTS .......................................................................................................... 6 2.1 INTERPHASE AND THE RESTRICTION POINT 6 2.2 DNA DAMAGE CHECKPOINTS 7 2.3 THE SPINDLE ASSEMBLY CHECKPOINT 8 3. CONTROL OF MITOSIS AND CYTOKINESIS BY POST-TRANSLATIONAL MODIFICATIONS ............ 8 3.1 UBIQUITINATION AND SUMOYLATION 9 3.1.1 Ubiquitination 9 3.1.2 SUMOylation 10 3.2 PHOSPHORYLATION 11 4. THE AURORA FAMILY OF PROTEIN KINASES .............................................................................. 13 4.1 AURORA KINASES FUNCTION AND LOCALIZATION 13 4.1.1 Aurora A: centrosomes and spindles 13 4.1.2 Aurora B: the catalytic member of the chromosomal passenger complex 14 4.1.3 Aurora C: only a male meiotic kinase? 15 4.2 REGULATION OF AURORA KINASES 15 4.2.1 Aurora A is regulated by Tpx2 16 4.2.2 Aurora B is regulated in a CPC-dependent manner 17 4.2.3 Aurora C, the unknown member of the family 18 4.3 MOUSE MODELS OF AURORA KINASES 18 4.4 AURORA KINASES AND CANCER 19 AIM OF THE WORK ................................................................................................................... 21 MATERIALS & METHODS ........................................................................................................ 23 1. GENETICALLY MODIFIED MOUSE MODELS ................................................................................. 23 iii Index 1.1 ANIMAL HOUSING 23 1.2 GENERATION OF MOUSE MODELS 23 1.2.1 Construction of targeting vectors 23 1.2.2 Generation of quimeras 24 1.2.3 Generation of Aurora B conditional, knock-in and null alleles 24 1.3 MOUSE CROSSES 25 1.4 MOUSE GENOTYPING 25 1.5 TREATMENTS IN LIVE ANIMALS 26 1.5.1 Wound healing 26 1.5.2 Hepatectomy 26 1.5.2 Tumor induction 26 2. HISTOLOGICAL AND IMMUNOHISTOCHEMICAL ANALYSIS ...................................................... 27 3. EMBRYOS ..................................................................................................................................... 27 3.1 EMBRYO CULTURE AND EXTRACTION 27 3.2 EMBRYO MICROINJECTION 29 4. CELL CULTURE ............................................................................................................................ 29 4.1 MEFS CULTURE, EXTRACTION AND INFECTION 29 4.2. HUMAN AND MOUSE CELL LINES 30 5. BIOCHEMICAL PROCEDURES ...................................................................................................... 30 5.1 RNA EXTRACTION AND REAL-TIME-PCR 30 5.2 DNA CLONING 31 5.4 IMMUNOFLUORESCENCE 31 5.4 X-GAL DETECTION IN EMBRYOS AND CELLS 31 5.5 PROTEIN EXTRACTION AND ANALYSIS 31 5.6 IMMUNOPRECIPITATION AND IN VITRO KINASE ASSAYS 32 RESULTS ........................................................................................................................................ 33 1. SUMO REGULATION OF AURORA B .......................................................................................... 33 1.1 AURORA B IS MODIFIED BY SUMO 33 1.1.1 Identification of a putative SUMOylation motif conserved in Aurora B 33 1.1.2 Aurora B mouse protein is SUMOylated at K207 residue 34 1.2. ECTOPIC EXPRESSION OF A SUMO-DEAD AURORA B PROTEIN INDUCES CELLULAR DEFECTS 35 iv Index 1.2.1 Lack of Aurora B SUMOylation at K207R induces polyploidy and nuclear defects 35 1.2.2 Lack of Aurora B SUMOylation compromises cell viability 36 1.3. AURORA B IS REGULATED BY SUMO 37 1.3.1 SUMOylation of Aurora B is important for correct mitotic progression and completion of cytokinesys 38 1.3.2 SUMO regulates Aurora B and Incenp centromeric localization 40 1.3.3 SUMOylation affects Aurora B function without reducing its kinase activity 41 1.3.4 An abnormal SUMOylation of Aurora B affects its oncogenic properties 42 2. DEVELOPMENT OF MOUSE MODELS ............................................................................................ 44 2.1 GENERATION OF A CONDITIONAL KNOCK OUT MOUSE MODEL 44 2.2 GENERATION OF A -GALACTOSIDASE KNOCK-IN MOUSE MODEL 44 2.3 GENERATION OF A TETRACYCLINE-INDUCIBLE MOUSE MODEL 47 3. PARTIAL IN VIVO INACTIVATION OF AURORA B ........................................................................ 50 3.1 LACK OF ONE ALLELE OF AURORA B DOES NOT RESULT IN MAJOR ALTERATIONS DURING MOUSE DEVELOPMENT 50 3.1.1 Aurora B heterozygous MEFs proliferate well in culture 50 3.1.2 Aurora B heterozygous mice are fertile although few of them develop hypospermia 50 3.2 LACK OF ONE ALLELE OF AURORA B RESULTS IN IMPAIRED PROLIFERATION AND SLIGHT PROTECTION AGAINST TUMOUR INDUCTION 51 3.2.1 Impaired in vivo proliferation in Aurkb(+/–) mice 52 3.2.2 Slight protection against tumour induction in Aurkb(+/–) mice 52 3.3 LACK OF ONE ALLELE FOR AURORA B INCREASES SUSCEPTIBILITY TO SPONTANEOUS TUMOUR DEVELOPMENT IN AGED MICE 54 4. LETHALITY AND COMPLEMENTATION BY AURORA C ............................................................... 55 4.1 GENETIC ABLATION OF AURORA B DOES NOT DISTURB EARLY EMBRYONIC DIVISIONS 55 4.1.1 Aurora B-deficient embryos progress normally to a blastocyst stage 55 4.1.2 Aurora B is expressed during first cell divisions 55 4.1.3 Histone H3 is properly phosphorylated and Incenp is correctly localized in early embryos lacking Aurora B 57 4.1.4 Aurora B-null blastocysts display normal size and cell number 58 4.2. GENETIC ABLATION OF AURORA B RESULTS IN MITOTIC ABERRATIONS AND LETHALITY AFTER IMPLANTATION 58 v Index 4.2.1 Histological examination of abnormal implanted Aurora B-null embryos 59 4.2.2 DNA damage and activation of the p53 pathway leads to apoptosis in implanted embryos lacking Aurora B 59 4.2.3 Aurora B-deficient cells arrest at prometaphase/metaphase with an active SAC 60 4.2.4 Embryos fully degenerate at E9.5 in the absence of Aurora B 60 4.3 GENETIC ABLATION OF AURORA B RESULTS IN ABNORMAL ADVANCED BLASTOCYSTS 61 4.3.1 Aurora B-null advanced blastocysts display an aberrant ICM surrounded with abnormal TGC 61 4.3.2 Lack of Aurora B prevents proper chromosome segregation in ES cells 62 4.3.3 Aurora B-deficient ES cells arrest at prometaphase with a functional SAC and mislocalization of the CPC 63 4.4. AURORA C COMPENSATES FOR AURORA B FUNCTION DURING EARLY EMBRYONIC DEVELOPMENT 65 4.4.1 Aurora C is highly expressed in early embryos 65 4.4.2 Inhibition of Aurora B/C results in a severe arrest in early embryos 66 4.4.3 Aurora C plays a crucial role in driving proper mitosis in early cell divisions 68 5. ESSENTIAL FUNCTIONS OF AURORA B IN G1/S PROGRESSION AND MITOSIS .......................... 68 5.1 AURORA B DEPLETION IN